One of the most remarkable advances in cell biology has been the continuing clarification of the signaling pathway by which mitogens, after binding on the cell surface receptors, transmit into the nucleus the signals which trigger de novo synthesis of DNA and mitosis. See, for example, R. J. Davis, J. Biol. Chem., 268, 14553 (1993). All components of this pathway are not identical in all cell types. However, the transmission of developmental and proliferative signals from the membrane to the nucleus requires the coordinated action of a diverse set of proteins. As shown in FIG. 1, the mitogen-signaling pathway typically includes receptor tyrosine kinases, the Src family of nonreceptor tyrosine kinases, Ras, Raf-1, MEK (MKK), MAPK, and RSK.
FIG. 1 also depicts two members of the Rho subclass of GTP-binding proteins, Rac and Cdc42, which are believed to target and activate the p21 -activated family of serine/threonine protein kinases called PAKs (p21-activated kinases). E. Manser et al., Nature, 367, 40 (1994).
The Raf-1 serine/threonine kinase serves as a control intermediate in these pathways, functioning both as a link between membrane-bound and cytoplasmic proteins and as a bridge connecting upstream tyrosine kinases such as EGF or PDGF receptors with downstream serine/threonine kinases. Both the essential role and the position of Raf-1 in many signaling pathways have been demonstrated from studies using deregulated and dominant inhibitory Raf-1 mutants in mammalian cells as well as from studies employing biochemical and genetic techniques in Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans (reviewed in T. M. Roberts, Nature, 360, 534 (1992)). In many cases, the activation of Raf-1 by receptors that stimulate cellular tyrosine phosphorylation is dependent on the activity of Ras, indicating that Ras functions upstream of Raf-1 (K. W. Wood et al., Cell, 68, 1041 (1992)). Upon activation, Raf-1 then phosphorylates and activates MEK (MKK), resulting in the propagation of the signal to downstream effectors, such as MAPK (mitogen-activated protein kinase) (C. M. Crews et al., Cell, 74, 215 (1993)).
Deregulation of the Ras signaling cascade can result in oncogenic cellular transformation through the constitutive activation of one or more downstream effectors. J. Bas, Cancer Res., 49, 4682 (1989); M. S. Marshall, FASEB J., 9, 134 (1995). Although a growing number of signaling proteins have been implicated as being subject to direct activation by Ras-GTP, the Raf serine/threonine kinases are considered to be the primary Ras effectors involved in the proliferation of animal cells. J. Avruch et al., Trends Biochem. Sci., 19, 279 (1994); C. M. Crews et al., cited above.
Normally localized in the cytosol in an inactive form, Raf-1 associates with Ras at the plasma membrane following growth factor-induced Ras guanine nucleotide exchange. S. Traverse et al., Oncogene, 8, 3175 (1993). Genetically engineered Raf-1, localized to the plasma membrane through posttranslational modification with myristate or isoprenoid, is active in the absence of Ras-GTP. This observation suggests that Ras functions primarily as a plasma membrane docking protein (G. Heidecker et al., Mol. Cell. Biol., 10, 2503 (1990); S. J. Leevers et al., Nature, 369, 411 (1994); D. Stokoe et al., Science, 264, 1463 (1994)). Once at the membrane, Raf-1 becomes catalytically activated through a complex and still largely undefined mechanism. These activation steps probably include both phosphorylation and a conformational change which relieves the inhibition imposed by the Raf-1 amino terminus (P. T. Dent et al., Science, 8, 1902 (1995); G. Heidecker et al., Adv. Cancer Res., 58, 53 (1992); D. K. Morrison, Molec. Reprod. Dev., 42, 507 (1996)). Lastly, lipid factors may bind to partially activated Raf-1, resulting in full activation. (See, Dent et al., cited above.)
Raf-1 activation requires phosphorylation, since treatment of active Raf-1 with protein phosphatases specific for either phosphoserine or phosphotyrosine results in loss of kinase activity (P. T. Dent et al., Science, 268, 1902 (1995); T. Jelinek et al., Mol. Cell. Biol., 16, 1027 (1996)). Raf-1 is predominantly phosphorylated in vivo on serines 43, 259, and 621 (D. K. Morrison, Mol. Reprod. Biol., 42, 507 (1996)). Phosphorylation of serines 43 and 259 has negative regulatory functions related to inhibition by protein kinase A and 14.3.3 binding. Serine 621 appears to have multiple roles. It is essential for Raf activation, but phosphorylation of this residue by protein kinase A or Raf-1 itself is associated with down regulation of Raf kinase activity (H. Mischak et al., Mol. Cell. Biol., 16, 5409 (1996)).
Potential inducible activators of Raf include protein kinase Cxcex1 (PKCxcex1) and Src family tyrosine kinases (W. Kolch et al., Nature, 364, 249 (1993)). PKCxcex1 phosphorylates Raf-1 on serine 499 (S499), which results in stimulation of catalytic activity in vitro. However, the in vitro activation of Raf-1 by membranes from cells transformed by Ras and Src does not require either S499 or PKCxcex1 (S. G. MacDonald et al., Mol. Cell. Biol., 13, 1615 (1993)). A more definitive regulatory role has been demonstrated for the Src-dependent tyrosine phosphorylation of Raf-1 residues 340 and 341 (J. R. Fabian et al., Mol. Cell. Biol., 13, 7120 (1993); R. Marais et al., EMBO J., 14, 3136 (1995)). Phosphorylation of tyrosine 340 and 341 (Y340 and Y341) strongly activates the kinase and transforming activities of full-length Raf-1. v-Src-dependent phosphorylation of Raf-1 Y340-Y341 has been shown to be dependent upon colocalization of Raf-1 with Ras-GTP at the plasma membrane. However, replacement of Y340 and Y341 with phenylalanine has no effect on the ability of a truncated Raf-1 CR3 fragment to transform fibroblasts, although v-Src-dependent phosphorylation dramatically increases the catalytic activity of this fragment. Furthermore, the physiological significance of Y340 and Y341 phosphorylation is uncertain since phosphotyrosine is difficult to detect on Raf-1 activated by growth factors in mammalian tissue culture.
Recently, it was demonstrated that phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-dependant activation and further biological signaling. B. Diaz et al., Mol. Cell. Biol., 17, 4509 (1997). The authors proposed that Raf-1 residues 338 to 341 constitute a unique site of coordinate serine and tyrosine phosphorylation and that this is one of several key events which occur only at the plasma membrane. A. J. King et al. reported isolation of a kinase activity capable of phosphorylating c-Raf-1 on serine 338/339 from rat spleen. The kinase was found to have a molecular weight of about 60 kD, and was insensitive to PKC and PKA inhibitors. Abstracts, 13th Ann. Meeting on Oncogenes, Frederick, Mo. (Jun. 18-21, 1997); Abstracts, 14th Ann. Meeting on Oncogenes, San Diego, Calif. (Jun. 24-27, 1998). However, the actual mechanism and consequences of these events remains unknown.
The present invention provides a method to determine the ability of a test compound to alter PAK3 activation of Raf-1 by contacting a polypeptide comprising the catalytic domain of PAK3 (p21-activated protein kinase) with the test compound in the presence of Raf-1 and measuring the extent of activation of Raf-1, by phosphorylation in vitro or in vivo. Because the activation of Raf-1 triggers downstream events in the MAPK signaling pathway that can lead to cellular proliferation, test compounds will typically be screened for their ability to inhibit PAK3 activation of Raf-1, i.e., to inhibit the ability of a PAK3 species to phosphorylate Raf-1 serine 338. The inhibition of PAK3 thus inhibits the resultant protein kinase activity of Raf-1 and, as a consequence, blocks activation of the whole downstream cascade, including the pivotal activation of MAPK. For example, see J. Wie et al., Science, 262, 1065 (1993) and J. Marx, Science, 262, 988 (1993).
Compounds that inhibit PAK3 activation of Raf-1 can modulate the mitogen cellular signaling pathway and provide treatments for neoplasia, lymphoproliferative disorders, arthritis, inflammation, autoimmune diseases, apoptosis and the like.
The term Raf-1 as used herein includes isolated and purified native and recombinant mammalian isoforms of Raf-1, as well as subunits (fragments), variants and analogs of Raf-1, such as Raf-1 CR3, that can be evaluated for phosphorylation and/or in growth/proliferation assays. Such variants, subunits and analogs conserve Ser 338 and, preferably, adjacent N-terminal amino acids.
Abo et al. (U. S. Pat. No. 5,605,825) have disclosed that identification of agents that modify the interaction of the human PAK1 species, hPAK65, with the rho-like p21 GTPases rac1 and CDC42H, may alter the activation of hPAK65. Abo et al. do not disclose or suggest a method to identify agents that can directly modify, i.e., inhibit the ability of PAK3 to phosphorylate and thus, to activate, Raf-1. It is not known if regulation of Raf-1 by PAK3 is dependant on either Cdc42 or Rac in vivo.